Three families of ligand-activated ion channels mediate synaptic communication between excitable cells in mammals. For pentameric channels related to nicotinic acetylcholine receptors and tetrameric channels like glutamate receptors, the pore-forming and gate regions have been studied extensively. In contrast, little is known about the structure of trimeric P2X receptor channels, a family of channels that are activated by ATP and serve crucial roles in neuronal signaling, pain transmission and inflammation. To identify the pore-forming and gate regions within P2X receptor channels, we introduced cysteine residues throughout the two transmembrane (TM) segments and studied their accessibility to thiol reactive compounds and ions. Our results show that the TM2 helix lines the central ion conduction pore, that the TM1 helix is positioned peripheral to TM2, and that the flow of ions is minimized in the closed state by a gate formed by the external region of TM2.